Tissue engineers seek to repair, replace, or regenerate damaged or diseased tissues by manipulating cells, creating artificial implants, or synthesizing laboratory-grown substitutes. One regenerative tissue engineering approach involves a process known as "tissue induction," whereby 21/2 and 3-dimensional polymer or mineral scaffolds without cells are implanted in a patient. With tissue induction, tissue generation occurs through ingrowth of surrounding tissue into the scaffold.
Another approach to tissue generation, known as "cell transplantation," involves seeding scaffolds with cells, cytokines, and other growth-related molecules, then culturing and implanting these constructs to induce the growth of new tissue. Cultured cells are infused in a biodegradable or non-biodegradable scaffold, which may be implanted directly in the patient, or may be placed in a bioreactor (in-vitro) to allow the cells to proliferate before the tissue is implanted in the patient. Alternatively, the cell-seeded scaffold may be directly implanted, in which case the patient's body acts as an in-vivo bioreactor. Once implanted, in-vivo cellular proliferation and, in the case of absorbable scaffolds, concomitant bio-absorption of the scaffold, proceeds.
In both types of tissue engineering, i.e., tissue induction and cell transplantation, the scaffold, whether or not bioabsorbable, must be biocompatible, such that it does not invoke an adverse immune response from, or result in toxicity to, the patient.
There exist numerous techniques for manufacturing scaffolds for tissue generation.
The techniques used are often dictated by the type of tissue ultimately being generated. One approach involves machining coraline hydroxyapatite to a desired shape. Another technique, known as "fiber bonding", involves preparing a mold in the shape of the desired scaffold and placing fibers, such as polyglycolic acid (PGA) into the mold and embedding the PGA fibers in a poly (L-lactic acid) (PLLA)/methylene chloride solution. The solvent is evaporated, and the PLLA-PGA composite is heated above the melting temperatures of both polymers. The PLLA is then removed by selective dissolution after cooling, leaving the PGA fibers physically joined at their cross-points without any surface or bulk of modifications and maintaining their initial diameter. Fiber bonding is useful for fabrication of thin scaffolds.
Another technique for manufacturing scaffolds is known as "solvent-casting and particulate-leaching." In this technique, sieved salt particles, such as sodium chloride crystals, are disbursed in a PLLA/chloroform solution which is then used to cast a membrane. After evaporating the solvent, the PLLA/salt composite membranes are heated above the PLLA melting temperature and then quenched or annealed by cooling at controlled rates to yield amorphous or semi-crystalline forms with regulated crystallinity. The salt particles are eventually leached out by selective dissolution to produce a porous polymer matrix.
Yet another technique used for constructing three-dimensional scaffolds is known as "melt molding", wherein a mixture of fine PLGA powder and gelatin microspheres is loaded in a Teflon.RTM. mold and heated above the glass-transition temperature of the polymer. The PLGA-gelatin composite is removed from the mold and gelatin microspheres are leached out by selective dissolution in distilled de-ionized water. Other scaffold manufacturing techniques include polymer/ceramic fiber composite foam processing, phase separation, and high-pressure processing.
Whichever type of scaffold is selected, the scaffold's purpose is to support cells, which, after being seeded into the scaffold, cling to the interstices of the scaffold and replicate, produce their own extra-cellular matrices, and organize into the target tissue.
Many of the above-described techniques require the use of severe heat or chemical treatment steps, which preclude seeding cells into the scaffold while it is being built, rather, require waiting until the entire scaffold has been constructed. This presents a challenge to seeding cells in three-dimensional scaffolds. None of the known scaffold materials allow growth of cells to a depth of greater than about 250 micrometers, which is a generally accepted practical limit on the depth to which cells and nutrients can diffuse into scaffolds having the desired porosities. Even if cells could be made to diffuse to greater depths, it is generally believed that to support cell growth and avoid or at least curtail apoptosis at these depths, the scaffold must also support some form of vasculature to promote angiogenesis; none of the scaffold fabrication techniques just discussed, however, allow for incorporation of blood vessels.
The cell transplantation approach has been used to produce bone, cartilage, liver, muscle, vessels, and skin analogues. In this approach, depicted in FIG. 1, cultured cells 100 are seeded into a three-dimensional, biodegradable scaffold 102. The resulting cellular construct, 104, is either cultured in-vivo, in a bioreactor 106, such as a broth medium placed in an incubator prior to implantation, 108, or is directly implanted in an animal or the patient 110. These synthesized tissues, however, replicate the histological composition and function of the desired tissue with varying degrees of accuracy. Complete organs, such as livers, and entire functioning groups, such as a vascularized bone with attached tendon and muscle, have yet to be demonstrated. These limitations are due, in part, to restrictions of the manufacturing methods presently used to fabricate three dimensional scaffolds, discussed above, which limit scaffold constructs to be homogeneous in microstructure, material composition, cell type and distribution.
Furthermore, except for monolayer structures, scaffolds have not been successfully developed for supporting heterogeneous selective cell seeding within the same scaffold.
Rather, current approaches use one type of scaffold material to promote one type of cell growth. For example, in the case of bone regeneration, optimal pore size for maximum tissue growth ranges from 200-400 .mu.m, and so scaffold materials with this pore size and having sufficient rigidity and biochemical properties to support loads are used for generating bone tissue. There exist, however, very few biological tissues, with skin and cartilage being possible exceptions, that can be accurately fabricated using only one type of cell supported on one type of scaffold. Most tissues are made up of numerous different cell types, each of which requires a different scaffold, possibly different growth factors, as well as different blood vessel architecture to ensure viability. For example, a limb is comprised of bone, muscle and tendon. Scaffolds such as hydroxyapatite, useful to support bone cells, are too brittle and non-pliable to act as scaffolding for muscle or tendons. Other heterogeneous tissues, such as liver and kidney, are even more complex. Most current scaffolds and tissue engineering techniques fail to permit heterogeneous tissues to be grown or provided with blood vessels. Furthermore, it has been suggested that cell growth factors should be present in concentration gradients in order to maximize cell development. Most current scaffold fabrication methods have no direct means of directly creating controlled gradients of growth factor, with the possible exception of 3-D printing.
The capability to create heterogeneous scaffold seeding systems would help to enable the regeneration of tissues, and collections of tissues, which exhibit more accurate histological structure and function than can be achieved with homogeneous constructs alone. This capability would permit different cells to be strategically placed in different regions of the scaffold, and each region could be composed of the optimal scaffold material and microstructure for organizing and stimulating the growth of cells in that region.
As is now apparent, one of the problems encountered by the tissue engineer is the need to incrementally build up scaffold material, selectively implant cells and growth factors throughout the entire scaffold and embed a vascular supply. A process known as solid freeform fabrication may offer some solutions. Solid freeform fabrication (SFF) refers to computer-aided-design and computer-aided-manufacturing (CAD/CAM) methodologies which have been used in industrial applications to quickly and automatically fabricate arbitrarily complex shapes. SFF approaches to creating scaffolds for tissue engineering are also being investigated.
SFF processes construct shapes by incremental material buildup and fusion of cross-sectional layers. In these approaches, illustrated in FIG. 2, a three-dimensional (3D) CAD model 112 is first decomposed, or "sliced", via an automatic process planner 114, into thin cross-sectional layer representations which are typically 0.004 to 0.020 inches thick. To build the physical shape, each layer is then selectively added or deposited and fused to the previous layer in an automated fabrication machine 116.
Rapid prototyping of design models, as discussed in Prinz, JTEC/WTEC Panel Report on Rapid prototyping in Europe and Japan (March 1997) incorporated in its entirety by reference herein, has proven useful for industrial applications. In such an approach, in order to support the structure as it is being built up, sacrificial layers may also be deposited when required, as illustrated in FIGS. 3 and 4. In one approach, each physical layer 118, which consists of the cross-section and a complementary shaped sacrificial layer, is deposited and fused to the previous layer as illustrated in FIG. 3, using one of several available deposition and fusion technologies. The sacrificial material 120 has two primary roles. First, it holds the part, analogous to a fixture in traditional fabrication techniques. Second, it serves as a substrate upon which "unconnected regions" 122 and overhanging features 124 can be deposited. The unconnected regions require this support since they are not joined with the main body until subsequent layers are deposited. Another use of sacrificial material is to form blind cavities 126 in the part. The sacrificial material is removed when the part is completely built up. As illustrated in FIG. 4, other building approaches only use support structures 128 where required, i.e., for the unconnected regions and steep overhanging features. These explicit support structures are typically deposited with the same material as the object being formed, but are drawn out in a semisolid fashion so that it is easy to remove these supports when the part is completed. For example, they may be deposited as thin wall structures.
There are several deposition and fusion processes currently in use or being developed for SFF. Some representative examples of SFF processes, which have also been investigated for tissue engineering applications, are illustrated in FIGS. 5 and 6. In the selective laser sintering process, depicted in FIG. 5, a layer of powdered material 130 is spread over the top surface of the growing structure 131. A CO.sub.2 laser 132 is then used to selectively scan the layer to fuse those areas defined by the geometry of the cross-section; this also fuses subsequent layers together. The laser beam is directed using computer-controlled mirrors 134 directed by the CAD data. The unfused material 136 remains in place as the support structure. After each layer is deposited, an elevator platform 138 lowers the part 131 by the thickness of the layer and the next layer of powder is deposited. When the shape is completely built up, the part is separated from the loose supporting powder. Subsequent heat treatment might also be required. Several types of materials have been investigated, including metals, ceramics, polymers, and polymer-coated metals and ceramics. While the materials which have been identified are primarily for industrial applications, the fabrication of hydroxyapatite scaffolds using selective laser sintering have been investigated using polymer-coated calcium phosphate powder. Additional post-processing, such as high temperature heating which bums out the binder, and then higher temperature sintering which fuses the powder together, is required to strengthen the part.
The three-dimensional printing (3D printing) process, depicted in FIG. 6, is another powder-based SFF approach used in industrial applications, but with potential use in forming scaffolds for engineered tissue. An ink-jet printing mechanism 140 scans the powder surface 142 and selectively injects a binder into the powder, which joins the powder together, into those areas defined by the geometry of the cross-section. As with selective laser sintering, an elevator platform 144 lowers the part 141 by the thickness of the layer and the next layer of powder is applied by the ink jet. When the shape 141 is completely built up, the part is separated from the loose supporting powder. The use of 3D printing for fabricating biomaterial structures out of bovine bone and biopolymers have also been used. The potential of 3D printing to intimately control the orientation and placement of porous channels and the overall shape of a device could make 3D printing well-suited for producing tissue generation devices. For example, microchannels to help support angiogenesis can be created in the scaffold using this technique. It would also be feasible to use the same or different microchannels to support cell growth via infused cells, harvest medium, growth factors, blood, etc. However, since the feature size achievable with 3D printing is about 100 .mu.m, a modified building strategy is required to fabricate highly porous, small diameter microstructures. To make porous polymer scaffolds, salt is used as the powder and the polymer is used as the binder. The salt, which acts as a porogen, is leached out of the completed shape by dissolving the completed shape in water, leaving a porous polymer scaffold.
"Membrane lamination" is another SFF-like technique used for constructing three-dimensional biodegradable polymeric foam scaffolds with precise anatomical shapes. First, a contour plot of the particular three-dimensional shape is prepared. Highly porous PLLA or PLGA membranes having the shapes of the contour are then manufactured using the solvent-casting and particulate-leaching technique. Adjacent membranes are bonded together by coating chloroform on their contacting surfaces. The final scaffold is thus formed by laminating the constituent membranes in the proper order to create the desired three-dimensional shape.
In addition to the capability to build up complex shapes, fabricating shapes by incremental material addition techniques allows multi-material structures to be created, by using selective deposition techniques, and prefabricated components to be embedded within the structures as they are being built up. For example, FIG. 7 depicts such a heterogeneous structure 150, with embedded components 152, multi-materials 154, and support materials 156. Such structures have been created for industrial applications, with a process called Shape Deposition Manufacturing (SDM).
As discussed previously, scaffold fabrication methods, whether conventional, SFF or SDM, typically involve heat or chemical actions which would destroy living cells or compromise the growth factors. With these methods, cells can only be added to the scaffolds after they have been prefabricated. Growth factors can also be added at or prior to this point. For a discussion of incorporating growth factors into scaffold materials, see Saltsman, "Growth-Factor Delivery in Tissue Engineering," MRS Bulletin, Nov. 1996, p. 62-65. Completed scaffolds are impregnated with cells by exposing them to cells suspended in liquid culture media; the cells then diffuse into and attach to the scaffolds. Assuming that the cells are given enough time to diffuse into and throughout the scaffolds, then there will be a uniform distribution of cells; selective placement of cells in three dimensional scaffolds, once formed, is not feasible. Diffusion rates also limit the practical size (thickness) of scaffolds, as most cells and associated nutrients cannot diffuse to a depth of greater than about 250 microns into the scaffold. Even if cells could diffuse to greater depths, the scaffold would require blood vessels to support the deeply seeded cells. In addition, the fabrication techniques discussed tend to produce scaffolds with uniform microstructure. And, while scaffolds can be composites of different materials, the composition, including growth factors, is uniform throughout.
There are exceptions to some of the specific limitations just cited. For example, some cell culture and transplantation techniques incorporate cells directly in collagen matrices before the collagen is molded into the final scaffold shape. Further, 3D printing techniques can create nonhomogeneous microstructure. One approach suggested for preparing three-dimensional synthetic tissues is described in Klebe, "Cytoscribing: A Method for Micropositioning Cells and the Construction of Two- and Three-Dimensional Synthetic Tissues," Experimental Cell Research 179 (1988) pp. 362-373. Klebe discusses the use of ink jet printing techniques to selectively deposit cell adhesion proteins on a substrate. This technique uses monolayers of cells growing on thin sheets of collagen. The sheets can be attached to one another by gluing them together with collagen.
Still, while all of the existing scaffold fabrication methods can be useful techniques for specific applications, a general method for creating large scale, heterogeneous three dimensional scaffold systems, capable of supporting 3-dimensional cell culture and vascularization does not exist.
Accordingly, a significant advance in the art could be realized by a three-dimensional cellular scaffold that avoids one or more of the aforementioned shortcomings of the prior art.